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A Theragnosis Probe Based on BSA/HSA Conjugated Biocompatible Fluorescent Silicon Nanomaterials for Simultaneous In Vitro Cholesterol Effluxing and Cellular Imaging of Macrophage Cells Shanka Walia, Anika Guliani, and AMITABHA ACHARYA ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01998 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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ACS Sustainable Chemistry & Engineering

A Theragnosis Probe Based on BSA/HSA Conjugated Biocompatible Fluorescent Silicon Nanomaterials for Simultaneous In Vitro Cholesterol Effluxing and Cellular Imaging of Macrophage Cells Shanka Walia,1,2 Anika Guliani1,2 and Amitabha Acharya*1,2 1

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P.) 176061, INDIA 2

Academy of Scientific & Innovative Research (AcSIR), New Delhi, INDIA

*

Author to whom the correspondence should be addressed, E-mail: [email protected]; [email protected]; Tel (off): +91-1894-233339; Extn. 397; Fax: +91-1894-230433

Title running head: Protein conjugated biocompatible fluorescent NPs as theragnosis probe for simultaneous in vitro cholesterol effluxing and cellular imaging of macrophage cells. KEYWORDS:

Silicon NPs, albumin proteins, cyto-compatibility, cholesterol effluxing,

confocal imaging

ACS Paragon Plus Environment


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ABSTRACT: Fluorescent silicon NPs (Si-NPs) and 3-mercaptopropionic acid coated CdS NPs (MPA-NPs) were prepared and conjugated with two different albumin proteins viz., BSA (B) and HSA (H). The absorption, fluorescence, FTIR, circular dichroism and gel electrophoresis studies confirmed the conjugation of proteins to NPs.

DPPH assay confirmed that the

conjugated proteins retained their functional activity even after chemical modifications. The sizes of Si-NPs by TEM were found to be ~8.7±2 nm whereas MPA-NPs showed individual particle size of ~4.6±1 nm.

The in vitro studies suggested that these NPs were highly

biocompatible. The potential of these protein conjugated NPs in cholesterol effluxing and fluorescence imaging was studied using two different macrophage cell lines viz., human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). Results suggested that HSA conjugated NPs showed better cholesterol effluxing ability and superior penetration towards these treated cells. Intracellular presence of Si-NPs was confirmed by confocal microscopic studies. All these studies unravelled the potential of Si-NPs as theragnosis probe for future biomedical applications.


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INTRODUCTION Different molecular imaging modalities has been used for diagnosis of diseases but unfortunately, all suffer from some drawbacks and need improvements.1 Nanotechnology considerably facilitates the development of molecular diagnostic probes.2 In the last few years, fluorescent nanoparticles (FNPs) with targeted biological applications have gained utmost attention.3,4 Fluorescence imaging plays an important role in clinical diagnosis due to their added advantages viz., ease of detection of events in real time, high sensitivity images with dynamic details and quantitative information at cellular/subcellular levels with high spatial resolution at nanometer range.5,6 Additionally, FNPs are widely used because these are brighter and stable than organic dyes and fluorescent proteins.7 The commonly used FNPs include inorganic NPs especially quantum dots (QDs) viz., CdSe, CdTe, CdS, PbS, ZnS, Ag2S, ZnO etc.2,8,9, rare earth oxides (iridium, ruthenium), lanthanide complexes, dye-doped silica NPs etc.10,11 In literature, Cd based FNPs has been extensively studied for bioimaging, but strong toxicity concerns restrict their clinical applications.8,11-13 In this respect there are some better substitutes as compared to Cd based QDs viz., carbon dots, metal nanoclusters, upconversion luminescent NPs and Si-NPs.4,14 Silicon (earth’s second most abundant material) NPs possess strong fluorescence emission, offers excellent biocompatibility (since Si degrades into silicic acid which is non toxic in nature and thus is easily excreted out from the body), with no or minimal inflammatory response in vivo.14-16 However smaller particles resulted from the fast biodegrdation of Si-NPs are generally recognized and scavenged as foreign particles by immune system, which is inevitably reflected in the decrease of fluorescence intensity of these NPs and restricts their use in long term bioimaging.14 Therefore to increase their bioavailability and



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retention time, it is indeed necessary to chemically modify their surface via oxidation, silanization, hydrosilylation, chemical conjugation etc.15,17 Albumin proteins viz., BSA (B) and HSA (H) share structural as well as ~75.52 % similarity in their amino acid sequence.18 Both of these albumin proteins can express their binding moieties for many ligands like various metabolites, hormones etc. though, to a different extent due to their difference in structural rigidity.18 It has also been known that these albumin proteins can play significant role in cholesterol effluxing in plasma.19 Further, surface coating of NPs with these albumin proteins may significantly increase the retention time of the corresponding NPs.14 Cholesterol, synthesized by liver or supplied in diet, is important in constituting cell membrane structure, production of hormones and shielding the nerves. Deposition of excess of cholesterol in cells or serum causes blocking of the arteries and is a major cause for cardiovascular disorders.20 Thus cholesterol content in the cells should be balanced otherwise it may lead to necrosis or apoptosis. Normally most of the cells can’t degrade cholesterol; however numbers of intracellular proteins are responsible for maintaining the required concentration of the same, inside and outside the cells.21 Here in this paper we have reported mild chemical synthetic procedure for the preparation of two different FNPs viz., silicon NPs (Si-NPs) and 3-mercaptopropionic acid coated CdS nanoparticles (MPA-NPs). Further to increase their efficacy in biomedical applications, these NPs were conjugated with BSA and HSA. Spectroscopic, microscopic and gel electrophoresis studies were done to characterize and confirm the conjugation of the respective proteins on NP surface. In vitro studies carried out with two different macrophage cells viz., human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC)


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suggested excellent biocompatibility, increased cholesterol effluxing ability for the protein conjugated NPs. The intracellular presence of the prepared Si-NPs was confirmed by confocal microscopic studies.

EXPERIMENTAL SECTION Materials. (3-Aminopropyl)triethoxysilane (APTES), (+)-sodium L-ascorbate, bovine serum albumin (BSA), human serum albumin (HSA), 3-mercaptopropionic acid (MPA), Nhydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC),

ammonium

methylenebisacrylamide,

persulfate, coomassie

tetramethylethylenediamine, brilliant

blue,

sodium

acrylamide,

dodecyl

sulfate,

N,N'glycerol,

bromophenol blue, cholesterol efflux assay kit (MAK192), sulforhodamine B (SRB), poly-Llysine, dialysis tubing cellulose membrane, Bradford reagent, 2,2-diphenyl-1-picryl hydrazyl (DPPH) and propidium iodide (PI) were purchased from Sigma-Aldrich. Amicon®ultra-15 centrifugal filter (cut off ~50,000 Da), trichloroacetic acid (TCA), glacial acetic acid (GAA) were purchased from Merck. Cell lines viz., human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cell (HUVEC), and their specific growth medias were purchased from Lonza, India.

Sodium sulfide flakes, cadmium chloride monohydrate,

glutathione (GSH), disodium hydrogen phosphate anhydrous, sodium dihydrogen phosphate monohydrate, NaCl, NaOH were purchased from SRL, India. Tri-sodium citrate and tris HCl were procured from SDFCL, India. Cell line namely BALB/C monocyte macrophage (J774) was obtained from National Centre for Cell Science (NCCS), Pune, India and its media RPMI



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1640 was procured from Gibco, Invitrogen. Sterile chamber slides with cover were purchased from Thermo Scientific. Initially, GSH coated CdS NPs (GSH-NPs) and corresponding conjugates of BSA and HSA (GB and G-H), MPA coated CdS NPs (MPA-NPs) and corresponding conjugates of BSA and HSA (M-B and M-H) and APTES coated Si NPs (Si-NPs) and corresponding conjugates of BSA and HSA (Si-B and Si-H) were synthesized and characterized. Synthesis of GSH-NPs, MPA-NPs and Si-NPs. Glutathione coated CdS nanoparticles (GSHNPs) were synthesized and characterized based on the report published elsewhere.8 MPA-NPs were prepared according to the already published literature report with modifications.22 Initially, 400 µl MPA was added to 40 ml CdCl2 (5 mM) and left for stirring at 160°C for 1 h under inert atmosphere. The pH of the solution was raised to 6.5 by dropwise addition of NaOH (1 mM). The reaction mixture was refluxed for next 30 min and then 40 ml Na2S (2 mM) solution was added dropwise and refluxed, while the color of the solution turned yellow. The reaction mixture was concentrated and then purified using dialysis membrane against Milli-Q water for 6 h. For Si-NPs preparation, 3 ml APTES was added to 12 ml preheated water and the solution was further heated at 80°C for 20 min.23 Next, 3.75 ml (+)-sodium L-ascorbate (0.1 M) solution was added to it and the reaction mixture was refluxed at 90°C for 2 h till the solution turned reddish brown. The cooled solution was preserved in an amber colored glass vial. Conjugation of BSA and HSA to the Prepared Fluorescent NPs. The conjugation of proteins viz., BSA and HSA to the NP surface was done by using EDC/NHS coupling reaction with some modifications.24 The activation of the protein surface was done by dissolving 4 mg/ml of


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respective proteins in 1 ml of EDC (0.1 M)/NHS (0.4 M), diluted in 10 mM NaOAc (pH 5) buffer in an amber colored vial and the solution was stirred for ~4 h. To these solutions, 1.0 ml of freshly prepared Si-NP solution was added and the reaction mixture was stirred under dark overnight. The as prepared protein conjugates viz., Si-B and Si-H were purified by using Amicon® filter of molecular weight cut off ~ 50,000 Da. Similar procedure was also followed for the preparation of M-B/M-H and G-B/G-H though the activation using EDC/NHS was done on MPA-NPs and GSH-NPs surface respectively. Characterization. The absorption and fluorescence spectra of prepared samples were recorded in NanoDrop 2000 UV-Vis spectrophotometer and Varian Cary Eclipse fluorescence spectrophotometer (ex/em slit width was 10/10 respectively).

Fourier transform infrared

spectroscopy (FTIR) studies were carried out in Agilent Cary 600 Series FTIR spectrometer. The hydrodynamic diameter and zeta potential values were measured using Zetasizer Nano ZS (Malvern Instruments). TEM and HRTEM images were obtained by Tecnai T20 twin, TEM 200 kV (FEI Netherlands). The confocal microscopy was done using confocal Carl Zeiss LSM 510 META. For UV-Vis and fluorescence spectroscopy, 3 ml of each of the samples were used whereas for DLS/Zeta studies 1 ml of each samples were used. For CD spectroscopy studies, 100 µl of each samples diluted in 1 ml water was used. Both TEM and HRTEM studies were performed by directly placing a drop of different NPs on carbon coated copper grids. Bradford and DPPH Assay. Protein estimation was done using Bradford assay whereas DPPH assay was performed to ascertain the functional activity of the conjugated proteins. SDS-PAGE. The protein conjugated fluorescent NP complexes were run for gel electrophoresis studies (SDS-PAGE) to check the binding of proteins on NP surface. The running time for gel



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electrophoresis was set for 6 h at 70 V and 50 mA. For gel studies, 10 µL aliquot of the individual samples were mixed with 10 µL loading buffer (50 % glycerol, 10 % SDS, 0.1 % bromophenol blue and tris-HCl at pH 6.8) and the mixture was casted into the gel wells and run along with the protein marker (12 % resolving gel, 4 % stacking gel). Coomassie brilliant blue was used for staining the gel for 1 h and then destained overnight. Circular Dichroism (CD) Spectroscopy. CD spectra of native BSA, HSA and NP conjugated BSA/HSA were recorded to observe the changes in the protein secondary and tertiary structures after chemical conjugation. The spectra were recorded between 190-340 nm. The helical content of free and bound BSA and HSA was calculated from the mean residue ellipticity (MRE), in terms of deg. cm2. dmol-1.

MRE =

θ 10rl (n)

where, θ is the observed CD (in milli-degrees), r = number of amino acid residues in proteins, l is the path length of the cell (in cm) and n is the molar concentration of albumin proteins.25,26

Cell Culture and Maintenance. Three different cell lines viz., HCAEC, HUVEC and J774 were maintained for in vitro studies. All the cells were incubated in a humidified incubator at 37°C with 5 % CO2 and the culture medium was changed as and when required.

Cytocompatibility Studies using SRB Assay. The cell viability was calculated using SRB assay.27 The macrophage cell lines viz., HCAEC and HUVEC were dispersed in 96 well plate with total cell density of 20,000 cells/well (100 µl/well) and maintained at 5 % CO2 and 37°C in 95 % air incubator for 12 h.28 Next, the cell media was removed and the cells were treated with three different concentrations viz., 32.5, 65 and 130 µg/ml (based on the concentrations of


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proteins attached on Si-B, Si-H, M-B and M-H, as estimated from Bradford assay). The control studies were carried out using similar concentration of native BSA and HSA. For Si-NPs and MPA-NPs, the concentration was adjusted by appropriate dilution. Cytocompatibilty studies were carried out at two different time intervals viz., 24 and 48 h. After the desired time duration, the cells were fixed with 10 % TCA and frozen at 4°C for 30 min and then SRB was added to stain the fixed cells for next 30 min. The excess dye was removed by washing the cells with 1 % acetic acid 5-6 times and then the cells were air dried. The protein bound dye was dissolved using 10 mM tris base. Finally, the plates were read using microplate reader at optical density of 510 nm and cell viability was calculated using the formula,

Cell viability (%) =

Absorbance of sample ×100 Absorbance of control

Cholesterol Efflux Assay. Cholesterol efflux assay was performed with both HCAEC and HUVEC to check the efficacy of the prepared NPs. The assay was performed on 96 well plate containing 1×105 cells/well. The adhered cells were first washed with RPMI media (without serum) and then the cells were treated with cholesterol effluxing reagents as per the instructions given in the kit (100 µl/well) and incubated for 16 h. The labeling reagent was not added for the control samples.

After 16 h, the cells were washed and treated with three different

concentrations viz., 32.5, 65 and 130 µg/ml of each sample viz., Si-B, Si-H, M-B, M-H, and BSA and HSA as controls and incubated for 2 and 4 h. Similar concentration of Si-NPs and MPA-NPs were used by adjusting the dilution. At the end of incubation, the supernatants were separated and their fluorescence was measured (ex/em = 482/515 nm). Finally, the cells were solubilized using 100 µl of cell lysis buffer and the fluorescence was measured (ex/em = 482/515 nm). The percentage of cholesterol efflux was calculated by the formula



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Cholestrol efflux (%) =

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Fm ×100 Fc

where Fm is fluorescence intensity of supernatant and Fc is the fluorescence intensity of cell lysate and supernatant.

Confocal Microscopy. The imaging studies were done by seeding HCAEC, HUVEC and J774 cells (6,000 cells/well) in 8 well chamber slides pretreated with poly-L-lysine and allowed to adhere for 24 h. After removing the media and subsequent washing with PBS, these cells were incubated with 100 µl of each sample (65 µg/ml) viz., Si-B, Si-H, M-B, M-H for 2 h. BSA, HSA, Si-NPs and MPA-NPs were used as controls. Then each of the samples was incubated with 200 µl of 1.5 mM PI for ~5 min followed by rinsing with 20 mM sodium chloride sodium citrate (SSC) buffer. Finally, the cells were imaged under laser scanning confocal microscope. The band pass for NPs was kept at 505-530 nm whereas for PI it was 560-615 nm.

RESULTS AND DISCUSSION The synthesis of GSH-NPs, MPA-NPs and Si-NPs and their protein conjugates were described in the experimental section. The chemical structure of APTES, MPA and GSH is depicted in Scheme 1.

O

O Si O

NH2

O

OH

O OH

(a)

O

SH

NH2

(b)

Scheme 1. Chemical structures of (a) APTES, (b) MPA and (c) GSH.


N H

(c)

SH H N O

O OH

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Spectroscopic Characterization of the Prepared Nanomaterials.

The absorption and

fluorescence spectra of GSH-NPs, MPA-NPs and Si-NPs were studied and the data were compiled in Figure 1. The absorption peaks were observed at ~440, 370 and 560 nm for GSHNPs, MPA-NPs and Si-NPs respectively (Figure 1A). Further, attachment of BSA and HSA on GSH-NPs, MPA-NPs and Si-NPs was also analyzed by spectrophotometric studies. Both BSA and HSA showed characteristic protein absorption band at ~277 and 275 nm respectively whereas protein conjugated NPs showed blue shift in the absorption band at ~261, 260 and 259 nm for G-B/G-H, M-B/M-H and Si-B/Si-H respectively (Supporting Information, Figure S1). Such change in absorption band might have resulted due to the conjugation of proteins on NP surface.29 Further, the characteristic absorption peaks of individual NPs were observed at ~440, 370 and 560 nm for G-B/G-H, M-B/M-H and Si-B/Si-H respectively, suggesting none or minimal interference of protein conjugation on individual NPs absorption profile (Supporting Information, Figure S1). The excitation (ex) wavelength vs. fluorescence intensity plot corresponding to each fluorescence emission (em) wavelength for individual NPs were summarized in Supporting Information, Figure S2.

Fluorescence spectrophotometric results suggested that GSH-NPs

showed maximum emission intensity of ~52 at ~527 nm when excited at 380 nm (Figure 1B, Supporting Information, Figure S2).

Similarly, MPA-NPs showed maximum fluorescence

intensity of ~754 at ~558 nm when excited at 360 nm. Si-NPs showed maximum fluorescence intensity of ~490 when excited at 440 nm with emission at ~528 nm. When BSA and HSA were excited at 280 nm, both showed emission at ~347 nm. The protein emission bands of conjugated NPs viz., G-B/G-H, M-B/M-H and Si-B/Si-H were observed at ~332/337, 343 and 341 nm respectively (Supporting Information, Figure S3 and Table S1). Such hypsochromic shift with



low fluorescence emission intensity in G-B/G-H might have resulted from the poor conjugation of both the proteins on GSH-NPs surface. When G-B/G-H, M-B/M-H and Si-B/Si-H were excited at their respective NP excitation wavelength, these showed emission peak at ~517, 558 and 512 nm respectively.

800

1.0

Fluorescence Intensity

A) 0.8

Absorbance

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0.6 0.4 0.2 0.0 300

400

500

600

700

800

Wavelength (nm)

700

B)

600 500 400 300 200 100 0 400

450

500

600

650

700

Wavelength (nm)

Figure 1. Absorption (A) and fluorescence (B) spectra of GSH-NP ( NP (

550

), MPA-NP (

) and Si-

) respectively.

UV Irradiation Studies. When all the samples were irradiated with 364 nm light, Si-NP, Si-B and Si-H showed blue fluorescence emission whereas MPA-NP showed orange emission, M-B and M-H showed light yellow emission (Figure 2). It was further observed that neither GSH-NP nor G-B/G-H responded to UV irradiation.


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Figure 2. Photographic images of the prepared fluorescent NPs and their protein conjugates under visible (top row) and UV (bottom row, excitation 364 nm) light. Glass vials arranged from left to right correspond to Si-NP, Si-B, Si-H, MPA-NP, M-B, M-H, GSH-NP, G-B and G-H, respectively.

Size and Surface Charge Analysis. To illustrate further the effects of proteins on NPs size and charge, DLS and zeta potential studies were performed. DLS and zeta results suggested size of GSH-NP, MPA-NP and Si-NP to be ~12.9±3, 93.8±15 and 4.8±1 nm and -36±6, -37±4 and 2.5±2 respectively (Supporting Information, Figure S4 and Table S2). The higher size of MPANPs might have resulted from the aggregation of individual MPA-NPs, which was later confirmed by HRTEM studies. It can be noticed that MPA-NPs showed maximum negative zeta potential value followed by GSH-NPs and Si-NPs, which can be correlated with the decreasing negative charge of individual ligands viz., MPA, GSH and APTES. The protein conjugated NPs viz., G-B/G-H, M-B/M-H and Si-B/Si-H suggested particle size to be ~6392/6483, 396±27/436±23 and 321±10/422±29 nm with corresponding zeta values in the range of ~-12.9±15/-19.9±14, -12.2±10/-21.4±12 and -6.5±10/-9.2±9 mV respectively. The increase in the particle sizes as well as change in the negative zeta potential values suggested protein conjugation on the NP surface. Further, the large particle sizes obtained for both G-B/G-H suggested possible sedimentation of both of these samples which in turn was supported by the precipitation observed for these samples (Supporting Information, Figure S5). Thus for all future studies only MPA-NPs and Si-NPs and their corresponding protein conjugates were used.



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Fluorescence Stability Studies. The stability of MPA-NPs/M-B/M-H and Si-NPs/Si-B/Si-H were analyzed by spectrophotometric studies over a period of 6 days (Supporting Information, Figure S6). Results suggested that MPA-NPs showed minimal change in fluorescence intensity as well as in peak position and symmetry whereas Si-NPs indicated decrease in fluorescence intensity. Protein conjugation on NP surface is expected to decrease the fluorescence intensity and this was observed for all the samples viz., M-B/M-H and Si-B/Si-H. Further, it may be mentioned that the comparative decrease in the relative fluorescence intensity plot suggested BSA/HSA conjugation resulted increased fluorescence stability for Si-NPs, which in turn is expected to enhance its utility in the biological milieu (Supporting Information, Figure S6).

Anchoring of proteins to the NP surface. FTIR analysis was carried out to confirm the conjugation of proteins on individual NP surface (Figure 3, Supporting Information, Table S3). MPA-NPs showed the presence of disulfide (-S-S-) peak at ~507.15 cm-1 whereas corresponding thiol (-SH) peak was absent at 2550-2600 cm-1, suggesting conjugation of MPA on CdS NP surface.

IR spectra of M-B and M-H showed both amide I (~1651.65 and 1650.76 cm-1

respectively, for C=O stretching) and amide II (1555.16 and 1558.18 cm-1 respectively, for N-H bending) peak which corresponds to protein conjugation on MPA-NP surface. Similarly, Si-NPs showed strong peaks at ~3487.68 and 1084.84 cm-1 corresponding to N-H (primary amine) and Si-OR stretching respectively. Further, Si-B and Si-H showed amide I peaks at ~1635.92 and 1644.64 cm-1 and amide II peaks at ~1552.45 and 1552.67 cm-1 respectively. All these results suggested successful conjugation of proteins on NP surface.30


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-1

Wavenumber (cm )

Figure 3. FTIR spectra of (A) MPA-NP and (D) Si-NP whereas (B), (C), (E) and (F) represents corresponding spectra for M-B, M-H, Si-B and Si-H respectively.

Protein Structural Integrity on NP Surface. The concentration of proteins on NP surface was estimated using Bradford assay31 (Supporting Information, Figure S7) and suggested ~1.3 mg/ml of protein presence in individual NP solution. The protein conjugation on NP surface was further confirmed by SDS-PAGE studies. Both BSA and HSA showed a thick band at ~66 kDa which was also observed for M-B/M-H and Si-B/Si-H samples, suggesting the presence of the respective proteins on the NP surface (Supporting Information, Figure S8A). The gel studies


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further indicated that the primary structure of the protein was retained even after chemical conjugation. The ability of the corresponding NPs to retain the secondary and tertiary structures of proteins was evaluated using CD spectroscopy (Supporting Information, Figure S8B). CD studies were carried out in the wavelength range of 190−340 nm where 200-260 nm represents far-UV range (for secondary conformational changes) and 250-340 nm signifies near-UV range (for tertiary conformational changes). All the samples viz., BSA, HSA, M-B/M-H and Si-B/Si-H showed the characteristic peak of α-helical conformation at ~ 207 and 223 nm (originated from n→π* and π→π* transitions respectively of the amide groups in the peptide bond), indicating protein secondary structures were retained after chemical conjugation.25, 32 Further CD plots of near-UV region resulted no significant differences for native proteins and NP conjugated proteins, suggesting that the tertiary structure of the respective proteins were preserved even after chemical conjugation.

Functional status of conjugated proteins. To verify whether the conjugated proteins retain their functional activities on NP surface, DPPH assay was performed.33 Increased anti-oxidant activity was observed for all the NP-protein conjugates suggesting retention of functional activities for the conjugated proteins (Supporting Information, Figure S9).

Morphological Characterization. All the NPs and their protein conjugated counterparts were characterized by TEM studies (Figure 4). The sizes of the individual Si-NPs were found to be ~ 8.7±2 nm whereas MPA-NPs showed particle size of ~4.6±1 nm. It may be noticed that for SiNPs mostly spherical and monodispersed particles were observed, though micrographs of MPANPs suggested aggregated and non-spherical structures.

When both of these NPs were

conjugated with BSA and HSA, larger size polydispersed particles of sizes ~26.9±5 (M-B), 49.5±4 (M-H), 194±25 (Si-B), 141±10 (Si-H) nm were observed where metallic NPs appeared as



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dark spots and the conjugated proteins can be visualized as thin film present around the NP surface. Further, HRTEM studies revealed lattice fringes corresponding to Cd and Si for protein conjugated NPs, thus suggesting presence of both the metals in the respective protein counterparts.


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A)

B)

D)

E)

F)

G)

H)

I)

J)

K)

L)

C)

Figure 4. TEM micrographs of (A) MPA-NP, (B) M-B (C) M-H, (G) Si-NP, (H) Si-B and (I) SiH. Corresponding HRTEM images are given in (D), (E), (F), (J), (K) and (L) respectively. Inset of (C) represents a closer look of M-H, with scale bar of 20 nm.



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To evaluate the biological efficacy of the prepared nanomaterials, cytocompatibility and cholesterol efflux assay were performed on two different macrophage cell lines viz., HCAEC and HUVEC whereas suitability of these prepared nanomaterials as molecular imaging agents were studied by confocal microscopy additionally on J774 (BALB/C monocyte macrophage) cells. The selection of these cell lines were made on the basis of the fact that macrophages participate in cholesterol homeostasis.

Further the over deposition of cholesterol causes

atherosclerosis in foam cell macrophages in the arterial walls and hence these cells can act as cholesterol donor.34-36

Cyto-compatibility Studies. Cyto-compatibility experiments were performed to evaluate the cellular tolerance limit of these prepared nanomaterials (Supporting Information, Figure S10). To check the cell viability, both the cells were treated with three different concentrations of NPs viz., 32.5, 65 and 130 µg/ml in triplicates and were incubated for 24 and 48 h. Studies suggested that all the samples showed excellent cyto-compatibility at both the time intervals and for both the cell lines.

Cholesterol Efflux Assay. Albumin proteins are known for their ability to act as reverse cholesterol transporter.19 The ability of the prepared nanomaterials to act as cholesterol efflux agent was tested against both the cells viz., HCAEC and HUVEC (Figure 5). Appropriate control studies were performed to monitor the effect of protein attachment on NP surface. From the graphs, it was evident that both BSA and HSA can act as cholesterol efflux agents, though at different extent for HCAEC (~66-85% and 92-98% for BSA and HSA respectively at 2 to 4 h) and HUVEC (~30-50% and 30-52% for BSA and HSA respectively at 2 to 4 h). Further it was observed that for HCAEC, both Si and MPA conjugated protein NPs showed an increase in the cholesterol effluxing ability to a marginal extent (~74-98% for Si-B/Si-H and ~84-98% for M-


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B/M-H), however no direct correlation could be established on protein concentration/time duration and cholesterol effluxing ability of the NPs. Interestingly, when HUVEC cells were treated with Si-B/Si-H and M-B/M-H, ~2 times increase in cholesterol effluxing ability was observed which was both concentration and time dependent i.e. higher cholesterol efflux was observed at higher concentration and longer time duration. Further it was found that Si-B/Si-H showed better cholesterol effluxing ability compared to M-B/M-H.

100

100

Figure 5. Plots for cholesterol efflux assay of (A) HCAEC and (B) HUVEC at 2 h ( (

M-H-2

M-H-3

Si-H-3 M-H-1

Si-H-2

H-3

Si-H-1

H-1 H-2

M-B-3

M-B-1

M-B-2

Si-B-3

B-3

M-H-2

M-H-3

Si-H-3 M-H-1

Si-H-2

0 H-3

0 Si-H-1

20

H-1 H-2

20

M-B-3

40

M-B-1

40

M-B-2

60

Si-B-3

60

B-3

80

Si-B-1 Si-B-2

80

Si-B-1 Si-B-2

B)

B-1 B-2

A)

B-1 B-2

Cholesterol efflux (%)

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) and 4 h

). The numerical 1, 2 and 3 in the figures corresponds to concentration of 32.5, 65 and 130

µg/ml respectively.

Cellular Imaging. To study the suitability of these NPs as bioimaging agents, three different cell lines viz., HCAEC, HUVEC and J774 were incubated with M-B/M-H and Si-B/Si-H for 2 h and imaged under confocal microscope along with the appropriate control NPs viz., BSA, HSA, MPA-NP and Si-NP (Figure 6 and Supporting Information, Figure S11). The green and red



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channel corresponds to NP and propidium iodide (PI) emission respectively whereas the last column reflects the merged images. It can be noticed that the red fluorescence response was observed for all the samples though the green fluorescence response was observed only for SiNP, Si-B and Si-H which was further supported by the corresponding merged images. Further, better fluorescence response was observed for HUVEC compared to HCAEC and amongst the three samples, green fluorescence intensity followed an order of Si-NP < Si-B < Si-H, suggesting more intracellular presence of silicon nanoparticles in case of Si-H. All these studies suggested that the prepared Si based nanomaterials can be used as molecular imaging systems for HCAEC and HUVEC.


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Si-NP

Si-B

Si-H

Si-NP

Si-B

Si-H



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Figure 6. Confocal microscopy images of different nanomaterials (65 µg/ml) treated HCAEC (rows 1 to 3) and HUVEC (rows 4 to 6) after 2 h. The first, second, third and fourth column corresponds to bright field, propidium iodide stained (PI, ex/em - 543/617 nm), nanoparticle treated (ex/em - 458/520 nm) and merged images, respectively.

CONCLUSION Three different nanomaterials and their BSA and HSA conjugates were synthesized viz., GSHNP/G-B/G-H, MPA-NP/M-B/M-H and Si-NP/Si-B/Si-H.

The absorption and fluorescence

studies confirmed the formation of NP as well as protein attachment. The protein attached nanomaterials showed blue shift in characteristic BSA/HSA peak, suggesting change in micro environment of the tryptophan residue of the corresponding proteins.37 In case of fluorescence studies, G-B/G-H did not respond strongly, reflecting poor conjugation of the proteins on GSHNP surface which was further proved by precipitation studies. Further it was observed that protein conjugation has resulted blue shift of the emission of the NPs. When irradiated with 364 nm light, Si-NPs and Si-B/Si-H as well as MPA-NPs and M-B/M-H emitted strongly whereas GSH-NPs/G-B/G-H did not respond. It should be mentioned here that CdS is a direct band gap semiconductor with band gap of 2.42 eV whereas Si is an indirect band gap semiconductor with band gap of 1.11 eV (at 320 K) and hence Si enjoys much longer excited state than direct band gap semiconductor.38 The DLS and zeta studies suggested increase in size as well as negative zeta potential of individual NPs after protein attachment. Further Si-NPs and Si-B/Si-H and MPA-NPs and M-B/M-H was found to be reasonably stable over a period of 6 days. The structural integrity and functional activity of the conjugated proteins on NP surface was


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confirmed by SDS-PAGE, CD and DPPH studies. TEM studies suggested the size of Si-NP to be ~8.7±2 nm whereas ~4.6±1 nm sizes were obtained for MPA-NP. Further, HRTEM and FTIR studies reflected that the metallic NPs are anchored on the large protein surface via chemical conjugation. Cytocompatibility studies suggested that MPA-NP showed reduced cell viability as compared to Si-NP, for both the cell lines, though protein conjugation on both NP surface increases their cytocompatibility. The cholesterol efflux assay suggested both the cells behave differently in presence of the nanomaterials. In case of HCAEC no direct relation could be made between NPs or their concentration and cholesterol effluxing percentage. But for HUVEC, protein conjugated nanomaterials showed better cholesterol effluxing with both time and concentration dependent manner. The differential cholesterol efflux ability of both the cells, can be attributed to the fact that HUVEC produces lower expression level of ABCA1 compared to HCAEC.39 It has also been reported that under similar experimental conditions, HCAEC produces a pattern of expression of chemokines distinct from that of HUVEC.40 The study suggested that NPs allows easy passage of protein inside cellular environment. Further it was also observed that both Si-B and Si-H showed better effluxing ability as compared to M-B and M-H whereas Si-H showed maximum efficiency. The confocal studies suggested non specific distribution of Si-NP and Si-B/Si-H in all the cells whereas more fluorescence response was observed in case of Si-H with HUVEC. This is in accordance with the earlier cholesterol effluxing results. All these results suggested that HSA allowed more penetration of NPs than BSA, in cells. Literature reports suggested that serum albumins with number of binding sites, acts as carriers for the transportation of different molecules/ligands through bloodstream. The binding cavity of HSA is more open-type whereas comparatively rigid BSA structure restricts its availability for incoming ligands41 and hence these two proteins behave differently in similar



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cellular environment. The present work reflects that with improved cholesterol effluxing ability and better fluorescence response in visible range, Si-NP as well as its protein conjugates might find their application as theragnosis probes for future biomedical applications.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Absorption and fluorescence, DLS and zeta potential, stability analysis, FTIR, Bradford assay, DPPH assay, cytocompatibility studies and confocal imaging.

Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS The authors are thankful to the Director, CSIR-IHBT for infrastructural facilities.

AA

acknowledges the financial support from CSIR, GOI in the form of BSC0213, BSC0112 and MLP0068. SW and AG acknowledge CSIR for project fellowship in the form of BSC0213 and BSC0112 respectively. AA acknowledges Dr. Y. S. Padwad for cell culture facility and Mr. Sourabh Soni for his assistance in cell culture work. The CSIR-IHBT communication number of this manuscript is 4050.


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For Table of Contents Use Only

A Theragnosis Probe Based on BSA/HSA Conjugated Biocompatible Fluorescent Silicon Nanomaterials for Simultaneous In Vitro Cholesterol Effluxing and Cellular Imaging of Macrophage Cells Shanka Walia,1,2 Anika Guliani1,2 and Amitabha Acharya*1,2 1

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P.) 176061, INDIA 2

Academy of Scientific & Innovative Research (AcSIR), New Delhi, INDIA

*

Author to whom the correspondence should be addressed, E-mail: [email protected]; [email protected]; Tel (off): +91-1894-233339; Extn. 397; Fax: +91-1894-230433

Synopsis Biocompatible protein conjugated fluorescent silicon NPs as theragnosis probe for simultaneous in vitro cholesterol effluxing and cellular imaging of macrophage cells.


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HSA ... - ACS Publications

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